Fig. 1

Heterogeneity of Hoxd mRNAs in single limb bud cells. a Scheme representing the organization of the Hoxd gene cluster and genes located nearby (bottom), together with the whole-mount in situ hybridization (WISH) pattern of the five most posterior Hoxd genes (Hoxd9 to Hoxd13) (top). The scheme illustrates both the similarity in the expression patterns and the progressive gain of expression, which correlates with the proximity of the target genes to the digit enhancers. Evx2 and Lnp are co-expressed with Hoxd genes in the developing digits, under the control of the same regulatory elements. b Fluorescent in situ hybridization of Hoxd13 mRNA on section reveals discrete expression pattern in the autopod of E12.5 mouse forelimb. c Single-cell double labelling of Hoxd13 and Hoxd11 mRNA from E12.5 autopod cells (up, schematic) by fluorescent hybridization followed by flow cytometry detection. The density plot (below) shows a high proportion of double negative cells. d Hoxd11 expression pattern revealed using a Hoxd11::GFP knock-in mouse strain, with high expression in digits and low expression in interdigital cells of autopod forelimbs, together with strong signals in zeugopod cells. e, f. Scatterplot profile from FACS to enrich for Hoxd11-positive cells using cells with high levels of GFP fluorescence (e, red) subsequently double-labelled for both Hoxd13 and Hoxd11 mRNAs. e Flow cytometry analysis from the GFP-positive cells from e are shown in a density contour plots where colors highlight the four population of cells expressing various levels of Hoxd11 and Hoxd13. g–i Double labelling of GFP (green, marker of Hoxd11-positive cells, g, h) and Hoxd13 (i, red, FISH) with DAPI (magenta) suggests four different combinations of Hoxd-positive cells: double positive for Hoxd13 and Hoxd11, single Hoxd13-positive, single Hoxd11-positive, and double negative for Hoxd13 and Hoxd11. j Histograms showing the correlation of expression where high levels of Hoxd13 are associated with high levels of Hoxd11 (two panels on the left). Higher levels of GFP are also observed in cells expressing at least one of the Hoxd genes (central panel). k Schematic of the four different combinations observed in f and h, i: double negative for Hoxd13 and Hoxd11 (purple), single Hoxd11-positive (green), single Hoxd13-positive (red), and double positive Hoxd13 and Hoxd11 (yellow)